PROJECT SUMMARY Yersinia pestis is the causative agent of human plague. Every year, approximately 2,000 cases of human plague occur, and in the past, several pandemics of plague have caused wide spread population loss. Y. pestis poses a threat to modern society due to its potential to be used as a bioterrorism agent, the absence of a FDA approved vaccine, and the possibility of antibiotic resistance. The identification of novel targets for therapeutic agents is critical for public health and safety. Consequently, Y. pestis is considered a Tier 1 Select Agent. For survival and virulence, Y. pestis must acquire transition metals, such as Fe, Zn, and Mn and overcome nutritional immunity, mechanisms in eukaryotic organisms that restrict nutrients from invading bacteria. During human plague, Yersinia pestis is able to overcome Fe limitation via production of the siderophore Yersiniabactin (Ybt). Recently, we identified an unexpected role for the Ybt system in the ability of Y. pestis to acquire Zn during infection. While the ZnuABC system contributes to in vitro growth in Zn-deficient media, a znu mutant is not attenuated in the mouse model of plague, unless the mutant also lacks genes involved in Ybt synthesis. These data suggest that Y. pestis uses two redundant Zn acquisition systems to cause plague. We hypothesize that Y. pestis produces a novel Ybt synthetase-dependent zincophore required for zinc acquisition and virulence. In Specific Aim 1, we will use a novel Tn-seq method to define genetic elements involved in the zincophore system. Genes that show a Zn phenotype will be validated through growth assays, trans-complementation, and biochemical experiments. In Specific Aim 2, we will determine the contribution of the Ybt synthetase-dependent zinc acquisition system to virulence by utilizing a hemochromatosis mouse model. The hemochromatosis mouse is defective in Fe nutritional immunity, which will allow us to distinguish between the contributions of Ybt synthetase-dependent Fe acquisition and Ybt synthetase-dependent Zn acquisition to Y. pestis virulence. In Specific Aim 3, we will determine the impact of calprotectin on Y. pestis virulence by using in vitro and in vivo methods. Completion of these Aims will define a novel secreted Zn acquisition system in Y. pestis. Our studies will be the first to determine the contribution of Zn acquisition to Y. pestis virulence in the mammalian host and the effect of calprotectin on plague infection. Furthermore, the involvement of conserved Ybt in this novel Zn acquisition demonstrates the significance of these studies to other bacterial pathogens.